Particuology 79 (2023) 27e34

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Particuology
journal homepage: www.elsevier.com/locate/partic

Positive and negative effects of graphite flake and monolayer

graphene oxide templates on protein crystallization
Zhichun Lin a, Wenqing Tian a, Yang Su b, Vikash Kumar Yadav c, Huaiyu Yang a, *
a
Department of Chemical Engineering, Loughborough University, Loughborough, LE11 3TU, UK
b
Institute of Materials Research, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
c
Human Sciences Research Centre, University of Derby, DE22 1GB, UK

a r t i c l e i n f o a b s t r a c t

Article history: Heterogeneous template-induced nucleation is a promising way to regulate protein crystallization events
Received 12 August 2022 and could be employed for purification processes and crystallographic studies. Protein crystallization
Received in revised form process with graphite and graphene oxide, as heterogeneous templates, were investigated. More than
10 October 2022
640 hanging drops with different concentrations of Lysozyme (30, 50, 70, 100 mg/mL) and NaCl (0.7, 0.9,
Accepted 27 October 2022
Available online 11 November 2022
1.1, 1.3, 1.5 M) were crystallised at 4
C with or without graphite/graphene oxide templates. The induction
times and crystallization process were observed under the microscope. The lysozyme in the solutions
with graphite flakes nucleated faster under all the conditions than the lysozyme with equal experimental
Keywords:
Graphite
conditions without templates. The crystals preferred to grow around the edge of graphite flakes than on
Graphene oxide the flat surfaces. In the droplets with monolayer graphene oxide, more crystals appeared around gra-
Protein crystallization phene oxide particles, and the faster or slower nucleation processes with templates were dependent on
Adsorption the lysozyme and NaCl concentrations. Graphene oxide templates strongly inhibited nucleation at high
Heterogeneous nucleation lysozyme concentrations but promoted nucleation at low lysozyme concentrations. Both heterogeneous
Template technology templates changed the crystal morphology and the crystallization kinetics. More crystals were observed
in the solution with graphite templatesthan with graphene oxide templates and without any template.
© 2022 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of
Sciences. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction nanoparticles (Hodzhaoglu et al., 2008)), soft templates (such as

Purification is an essential step in the protein production pro-
cess, especially in biopharmaceuticals, which can account for more
than half of the total production cost (Saikumar et al., 1998) in the
biomanufacturing process. Proteins are highly fragile and suscep-
tible to temperature, pH and other external factors (Chen et al.,
2020), therefore a suitable purification method is significant. Pro-
tein crystallization technology is an economic and effective method
in the purification process of protein products, which can obtain
single crystals with high purity (Hodzhaoglu et al., 2008; Saikumar
et al., 1998). Protein crystallization still has many challenges, such
as slow nucleation and crystal growth rates. In addition to the
traditional methods of reducing solution supersaturation such as
cooling, evaporation and extraction, heterogeneous templates, such
as hard templates (such as silica (Chen et al., 2020) and gold
* Corresponding author.
E-mail address: H.yang3@lboro.ac.uk (H. Yang).
bubbles (Tian et al., 2021), live cells (Sun et al., 2022), and DNA
(Zhang et al., 2018)) have also been reported in improving the
crystal structure (McPherson & Shlichta, 1988) and increasing the
crystal numbers (Tian et al., 2021). Heterogeneous nucleation,
occurring on the heterogeneous surfaces in solution, allows a lower
nucleation free energy and a faster nucleation rate than that of
homogeneous nucleation (Jara et al., 2019; Tanaka & Iijima, 2014). A
variety of carbon nanomaterials have also been proved to expand
the metastable range of crystallization (Govada et al., 2016). The
crystal form at the heterogeneous interface can be changed
(Tsekova et al., 2012).
Carbon-based materials, such as graphene (Tanaka & Iijima,
2014), carbon black and carbon nanotubes (Sun et al., 2022), have
been applied in protein crystallization as templates to accelerate
the crystallization process. Graphite material has attracted re-
searchers’ attention because of its special structure. Graphite is only
composed of sp2 hybrid carbon atoms (Jara et al., 2019) and has
plenty of p electrons. It is often used as electrode material and
lubricant. Graphene oxide (GO) has covalently connected

https://doi.org/10.1016/j.partic.2022.10.014
1674-2001/© 2022 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Z. Lin, W. Tian, Y. Su et al.
hydrophilic and hydrophobic areas, which makes it an amphiphilic
material (Wang et al., 2022). Surface hydrophilicity leads to the
wetting of solid nucleolus surface, facilitating the nucleation. It was
reported that GO templates can promote crystallization of catalase
due to the synergistic interaction between GO and cations (Zou
et al., 2019). Enzyme and protein molecules can also be directly
immobilized on GO because of the functional groups on its surface
and the partial graphitic structure (Zhang et al., 2013).
Lysozyme is a cationic protein in the human respiratory tract
and is widely distributed in animals, plants, and microorganisms
(Grosfils & Lutsko, 2021). As a natural bactericidal, lysozyme can
degrade the cell membrane to destroy and kill bacteria. Lysozyme is
a small 129 amino acid long monomeric protein stabilized by four
intramolecular disulfide bonds. It is a non-toxic, harmless, and safe
protease (Liu et al., 2007). The enzyme is active over a broad pH
range (6.0e9.0). At pH 6.2, maximal activity is observed over a
wider range of ionic strengths (0.02e0.100 M). It was reported that
a-helices appear to be somewhat more robust than b-sheets upon
adsorption on graphite and GO would significantly changes sec-
ondary and tertiary structures of the protein, forming a more stable
hybrid complex. Hydrogen bonding interaction was also reported
between lysozyme and carbon nanotubes (Li et al., 2022; Raffaini &
Ganazzoli, 2010; Wang et al., 2022). However, the influence of
graphite and GO templates on the protein crystallization and the
mechanism are not fully understood.
In this study, lysozyme was chosen as a model protein due to its
robust crystallization process and simple crystallization conditions
(Grzesiak & Matzger, 2008). Most of the crystallization work with
heterogeneous templates were reported to have positive influence
on the crystallization, such as enhancing the nucleation by
decreasing the nucleation free energy. In this research, graphite
flake and monolayer graphene oxide were chosen as heteroge-
neous templates for lysozyme nucleation. Effects of two types of
templates have been investigated with lysozyme concentrations
from 30 to 100 mg/mL and precipitant (NaCl) concentrations from
0.7 to 1.5 M in an incubator at 4
C. The induction time and
nucleation number were determined, and the crystal shape and
surfaces were observed by SEM and UV microscope. However, in
this work, the positive and negative influences were observed with
templates dependent on the crystallization conditions.
2. Experimental
2.1. Materials
Hen egg white lysozyme (around 70,000 units per mg), Sodium
acetate (assay >99%), acetic acid (>99.5%) and NaCl (>99.5%) were
purchased from Sigma-Aldrich. The graphite flake with a nominal
diameter of few hundreds of micrometers was purchased from
Sigma-Aldrich. Buffer solutions were prepared by adding sodium
acetate solid powder into 0.1 M acetic acid, adjusting the pH to 4.2.
2.2. Methods
Lysozyme solutions with concentrations of 30, 50, 70, and
100 mg/mL, were prepared in the buffer solution. The protein
concentration has been verified by measuring the absorbance in
the UVevis spectrophotometer (Thermo Scientific™ Nanodrop
One©). The precipitant was the buffer solution with NaCl concen-
trations of 0.7, 0.9, 1.1, 1.3, and 1.5 M All solutions were filtered
through a 0.2 mm cellulose acetate filter (Whatman) and stored in
the incubator at 4
C. The monolayer graphene oxide (diameter
<5 mm and thickness ~1 nm) was prepared by a modified Hummers’
method followed with mild sonication and centrifugation (Zhao
et al., 2010). Zeta potential of GO templates were about 42 mV.
28
Particuology 79 (2023) 27e34
The graphite flake and monolayer graphene oxide samples were
characterised with SEM (JSM-7100F), EDS (Oxford Instruments)
and AFM (Bruker Dimension Icon) for monolayer graphene oxide.
1 mL of precipitant was added into each well in a 24-well plate
(ThermoScientific Nunclon Delta Surface). The hanging droplets
shown as Fig. 1(a) mixed with 1.5 mL lysozyme solution with 1.5 mL
precipitation solution as Fig. 1(b). For the crystallization with
templates the graphite flake (200e500 mm) or the GO suspension
(1e5 mm) were premixed with percipient solution shown as
Fig. 1(c) and (d) respectively, before the precipitation solution
mixed with the lysozyme solution. There were four droplets on
each cover slide with and without template in the solution. The
coverslip was carefully inverted above the reservoir and sealed it
tightly with grease to ensure the closure of the system. The hanging
droplets were kept in an incubator (VWR INCU-Line 150R) at 4
C
and observed under optical microscope (GT Vision GTC-20) with
observation intervals of 30 min in the first 2 h and every hour
subsequently. The time to observe first crystal in one droplet was
recorded, and the average nucleation time of the droplets (average
12 droplets for each condition) under same experimental condition
with and without templates was compared. The droplets with
graphite templates and crystals at the end of experiments were
observed under UV microscope (Nikon Eclipse TE-300).
The solution of hanging droplets with lysozyme crystals and
templates was transferred on the silicon wafer carrier. A filter paper
was used to absorb the droplet solution on the silicon wafer. The
remaining crystals and templates on the silicon wafer were air-
dried and coated with Pd, which was observed by scanning elec-
tron microscope (JSM-7100F).
3. Results and discussions
Figs. 2 and 3 show a trend that with the increase of the NaCl
concentrations or lysozyme concentrations, nucleation time
reduced, which was consistent with literatures (Lin et al., 2017).
Fig. 2 shows that the nucleation times in the droplets with graphite
templates were all shorter (with average of 57% less) than those
without under equal conditions of salt and protein concentrations.
The nucleation rate could be accelerated up to double with graphite
templates in the droplet. Comparing the droplets with same lyso-
zyme concentration at 30 mg/mL, the difference of nucleation rates
increased with increase in the NaCl concentrations. Comparing the
solutions with same NaCl concentration at 0.7 mg/mL, the differ-
ence of nucleation times increased with increase in the lysozyme
concentrations. The influence (absolute period difference) was
more significant at low protein and salt concentrations, up to 5 h.
Fig. 1. Hanging drop crystallization method (a), without templates (b), with graphite
templates (c), and GO templates (d).
Z. Lin, W. Tian, Y. Su et al. Particuology 79 (2023) 27e34

Fig. 2. (a) Nucleation time of lysozyme in the solution with Graphite templates (black columns) and without (yellow columns), with lysozyme concentration of 30e100 mg/mL and
NaCl concentration of 0.7e1.5 M. (b) The percentage of nucleation time reduced by adding graphite templates in the droplets than those without template. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. (a) Nucleation time of lysozyme in the solution with GO templates (black columns) and without (yellow columns), with lysozyme concentration of 30e100 mg/mL and NaCl
concentration of 0.7e1.5 M. (b) The percentage of nucleation time reduced by adding GO templates in the droplets than those without template. (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)

When protein and salt concentrations were higher, the nucleation
times all decreased so the absolute period differences were not
obvious, but the difference of nucleation time in percentage could
still be high as over 70% under most conditions with high
supersaturations.
Fig. 3 shows complex influences of graphene oxide on the
nucleation process, dependent on the concentrations of lysozyme
and NaCl. With concentration of lysozyme 30 mg/mL in the solu-
tion, nucleation was promoted by adding GO templates. However,
with higher concentrations of lysozyme in the droplets, the
nucleation was hindered in most conditions. In most of the cases,
with same concentration of NaCl in the droplet, the nucleation time
was shorter with GO templates than those without at low con-
centration of lysozyme. Interestingly, with higher concentration of
lysozyme the differences of nucleation time became similar and
then the trend turned opposite as the nucleation rate was slower in
equal concentration of NaCl. Only when salt concentration was
1.1 M, GO promoted nucleation with all range of lysozyme con-
centrations. In other cases, the effect become opposite when lyso-
zyme concentrations were over 50 mg/mL. It is noted that with
0.9 M and 1.5 M NaCl in the droplets, GO templates hinder the
nucleation rates in the solution with all range of lysozyme con-
centrations above 30 mg/mL.
Graphite and GO templates lead to more crystals in 90% of the
droplets than those with equal condition without templates, shown
in Fig. 4. There were more crystals inside the droplets with gra-
phene oxide template in the droplets than those without during the
same crystallization time. Under high protein concentrations, more
than 70% of droplets with graphite templates resulted in more
crystals. At high NaCl concentration, there were up to 100 times
more crystals in the droplets with graphene template than those
without templates at the equal crystallization time.
The impurity contents in GO and graphite templates were
determined by energy-dispersive X-ray spectroscopies. The results
shown in Fig. 4(d), (e) and (f) indicate the impurities in both GO and
graphite template samples were very limited, only about 3%, with
similar quantity for each element of Na, K, Mn. Therefore, the huge
differences of the crystal numbers in the droplets with the GO and
graphite templates were not linked to the difference of the impu-
rities in these templates.
In almost all the droplets with graphite templates, there were
many crystals observed on the edge of the graphite flakes. In 40% of
droplets with graphite templates, small crystals form a crystal layer,
like a thick film, covering all the edges of the graphite flakes shown
in Fig. 5(a). With high salt and protein concentration, more than
60% of droplets presented this phenomenon on the graphite tem-
plates. The small crystals fast nucleated and agglomerated on the
edge of the graphite templates, and then the crystal film grew
thicker. The crystal not attached on the graphite templates all had
tetragonal shape. Similar phenomena have been reported, such as
fat crystals starting nucleation on the surface of graphite and
forming thin plate crystals (Sun et al., 2014). Fig. 5(c) shows the
protein crystals observed under UV microscope. The protein crys-
tals on the graphite template, which were not able to be observed

29
Z. Lin, W. Tian, Y. Su et al. Particuology 79 (2023) 27e34

Fig. 4. Optical microscope images of crystals in the droplets without template (a), with GO templates (b), and graphite templates (c) in the condition of pH ¼ 4.2, 1.1 M NaCl, and
70 mg/mL lysozyme at 1 h. (d) Elemental compositions of GO and graphite templates. Energy-dispersive X-ray spectroscopies for GO (e) and graphite templates (f).

Fig. 5. Optical microscope image (a), SEM image (b), and UV microscope image (c) of the lysozyme crystals from the droplets with graphite template.

under optical microscope, were highlighted under UV microscope
due to the biomolecules fluoresce when illuminated with UV ra-
diation. There were much more crystals grown closed to the edge of
the graphite flakes than those on the flat sheet at a distance from
the edge areas, which was consistent with the SEM images shown
in Fig. 5(b).
It is obvious that the lysozyme preferred to nucleate and grow at
the edge of graphite flakes rather than on the flat sheet side. The
possible reason is that the edges of graphite was rough, providing
more heterogeneous sites for the nucleation, which was in agree-
ment with the previous reports that the roughness of material lead
to faster nucleation in different systems (Grosfils & Lutsko, 2021;
Liu et al., 2007). The bonds on the edge of graphite flake with CeC
bonds were different with the interactions on the flat sheet side,
which were van der Waals bonding. The flat sheet surface and the
edge were both hydrophobic, but the breakage of CeC bonds may

30
Z. Lin, W. Tian, Y. Su et al.
be more attractive to protein molecules, contributing to a favour-
able nucleation cite for lysozyme crystallization. The preference of
the nucleation cites can be used to design the selective crystalli-
zation process and the interactions between the edge of the
graphite flake with the proteins can be potentially used to func-
tionalize the graphite edges and surface.
The lysozyme preferred to nucleate around GO templates.
Fig. 6(a) shows that with low concentration of the lysozyme and
NaCl, the GO templates settled in the middle region of the droplets,
where lysozyme nucleated first, and the most lysozyme crystals
were observed. The same phenomena were consistent in most of
the droplets with low concentration of the lysozyme or NaCl, due to
sufficient time (more than 5 h) for settling down of the GO tem-
plates at low supersaturation before nucleation. With high super-
saturation, the nucleation occurred before the GO templates settled
down in the middle region of the droplet, and the crystals were
more uniformly distributed in the droplets, shown in Fig. 6(b).
GO templates attracted lysozyme molecules due to the elec-
trostatic interaction and large amount of oxygen functional groups
(Jewel et al., 2015). The adsorption of lysozyme molecules on GO
template was reported, which can be reversed by adding alkali
(Grzesiak & Matzger, 2008), indicating the influence of the pH on
the electrostatic interactions. The properties of lysozyme crystals
Fig. 6. Lysozyme crystals from droplets with GO templates in the condition of 0.7 M NaCl and 50 mg/mL lysozyme: microscope image (a) and SEM image of a single crystal example
(c), and in the condition of 1.5 M NaCl and 100 mg/mL lysozyme: microscope image (b) and SEM image of a single crystal example (d). SEM (e) and AFM (f) images of the monolayer
GO templates with height profile of GO sample (i) and (ii).
31
Particuology 79 (2023) 27e34
surface with GO templates in the solution were rough with many
defects, shown in Fig. 6. It is interesting to observe that at low su-
persaturation, the GO templates settled in the middle areas of the
droplets (due to long induction time), the crystals had highly rough
and potholed surface, shown in Fig. 6(b). However, the crystals
formed in the outer areas still had good smooth surface as normal
lysozyme crystals obtained without templates, indicating the rough
surface is influenced much largely by the growth step than the
nucleation step.
While at high supersaturation, the nucleation occurred fast and
crystal grew with GO templates around, and therefore, the defects
appeared on nearly all the crystals surfaces. Comparing Fig. 6(d)
and (e), it was confirmed that the defect in Fig. 6(d) was the GO
templates enfolded inside the crystals. Fig. 6(e) and (f), SEM and
AFM images of GO templates, in show the sizes of GO templates
were all below 5 mm. Fig. 6(g) and (h) show the height profiles of
two GO template samples, and these two GO nanosheets had an
average thickness between 1 and 1.5 nm, suggesting the monolayer
nature of the graphene oxide templates. Except of the enfolded GO
templates inside and on the surface of the lysozyme crystal, most of
the crystal surface kept smooth and the crystals had normal and
tetragonal shape.
Z. Lin, W. Tian, Y. Su et al.
The density of the GO templates in Fig. 6(a) has more concen-
trated distribution (concentrated in the middle of the droplet) than
that in Fig. 6(b) (distributed in the whole droplet), and more tem-
plates around the crystal during the crystal growth led to the
rougher crystal surface in Fig. 6(a) and (c). The defects of lysozyme
were also reported with nano graphite templates, and the nano
graphite have been grown included inside the lysozyme, without
change the structure of the molecular unit cell of lysozyme crystal
(Gully et al., 2012; Kang et al., 2022). The bioavailability of these
crystals was not determined, due to insufficient amount of crystals
obtained in the hanging drop experiments. Nearly all of the crystals
obtained in this work were tetragonal shape (Grzesiak & Matzger,
2008), expected to be same form and conformation.
Two interesting phenomena were observed, first is the unex-
pected favourable effect of GO template only at 1.1 M salt solution.
Overall, there was an increasing positive influences of graphite
template (shorter induction time in the droplets with templates
than those without template) on nucleation with NaCl concentra-
tions from 0.7 M to 1.5 M, shown in Figs. 3 and 7. Lysozyme mol-
ecules with buffer pH of 4.2 have the isoelectric point of 11 [30],
indicating a positive charge in the droplets. Graphite with negative
charges (Li et al., 2014) in the droplets lead to electrostatic in-
teractions with lysozyme, contributing to the adsorption of the
protein molecules on the surface, especially the edge of the
graphite flake. Lysozyme has a strong initial adsorption on the
hydrophobic graphene surface and protein chains are usually ar-
ranged in parallel to each other to optimize the intramolecular
potential energy, resulting in a very stable final state (Gully et al.,
2012). The adsorption process was reported to be irreversible [33].
However, GO templates inhibited nucleation (longer induction
time in the droplets with templates than those without template)
in most of the conditions, requiring longer induction times
compared with that without templates. GO templates have
adsorption ability to the macromolecules (Mücksch & Urbassek,
2016; Sun et al., 2014). As adsorption of molecules would in-
crease the local concentration of the solute and increase the
probability of molecule collision (Chen et al., 2020), leading to a
faster nucleation. However, a much stronger interactions between
the GO templates and protein molecules, due to the function
groups, C]O and eOH, would reduce the mobility of the protein
molecules on the template surface (Chaudhary et al., 2021), in-
crease the difficulty level of forming the correct arrangements for
forming the nuclei, which probably be a reason for the inhibiting
effects of GO templates on nucleation. It is surprising to observe the
Fig. 7. Induction time changes (average value of percentage differences) in groups with equal concentration of NaCl (a) or lysozyme (b), comparing the droplets with templates to
the droplets without templates.
32
Particuology 79 (2023) 27e34
positive effect only with 1.1 M salt concentration, shown in Fig. 7(a)
and more experiments were repeated to confirm that the phe-
nomenon did not randomly appear. A similar trend was reported,
that with increase of the NaCl or MgCl2 concentration, increase or
decrease influences for crystallization of protein catalase in the
solution with GO templates (Zou et al., 2019). The same GO tem-
plates were used in all the experimental conditions with different
salt concentrations. The elementary impurities existed in all the
experiments, but they were very limited and the concentrations
were similar in all the experiments. One of the possible reasons can
be the special electrostatic interactions at 1.1M salts, and the 3D
structure of protein molecules adjusted by the free cations in so-
lution is coincidentally similar to the crystalline structure (Zhao
et al., 2010), leading to a favourable effort on nucleation. But the
mechanism is not fully understood, and the literature and the
current work revealled the complicated influence by combination
of the influence of ion, GO, and pH, which needs further in-
vestigations. Fig. 7(b) shows the difference of the influence be-
tween two templates increased with increase in the concentration
of lysozyme concentrations. With graphite templates, the induction
times all increased in the droplets compared with those without
templates, and the influence tended to increase with increase in the
lysozyme concentration. With GO templates, the stronger negative
effects were observed with high concentration of lysozyme, and the
effects tended to be weaker with decrease of the lysozyme con-
centration. It is noticed that at very low concentration, the GO
templates had some extent of positive influences. This work
demonstrated the complicated interactions between the graphite
and GO templates with lysozyme and salts in the solution, more
protein systems are worth to be investigated in the future for better
understanding these interactions.
The second interesting observation was a special patten of
crystals appearing in the droplets with the graphite template. In
almost all cases without templates and with GO templates, the
crystal located in the droplets randomly distributed and overall
uniformly around the whole droplet. It was noted that in many
cases with graphite templates the distribution of crystal in the
droplets was not random. Fig. 8 shows that there were significantly
more crystals on one side of the template in the droplet than in
other regions.
It was observed in over 60% droplets with a nucleation time
between 0.5 and 3 h had such special patten. In the droplets with
very low and very high supersaturation (very short and very long
nucleation time), there were limited number of crystals and very
Z. Lin, W. Tian, Y. Su et al.
Fig. 8. Optical microscope images of ununiformly distributed crystals in the droplets with one graphite template (a), two graphite templates (b) and without template (d) in the
condition of 1.3 M NaCl, 50 mg/mL lysozyme, with graphite template (c) in the condition of 1.1 M NaCl, 100 mg/mL lysozyme.
crowed crystals around the droplets, respectively, which was
difficult to distinguish such pattern. In order to understand the
phenomena, we tried to put two graphite templates in the droplets,
however, the probability of this phenomenon was not increased,
and no obvious regulation has been found, as shown in Fig. 8(b).
The proposed mechanism was that graphite could probably affect
the distribution of charge and ions in the solution, influence the
local supersaturation of the droplets, inducing preferable locations
for nucleation. The mechanism needs to be further investigated,
which can be used to design the crystallization locations and con-
trol the protein crystallization process.
4. Conclusion
The positive effects of graphite templates and the concentration
linked negative effects of graphene oxide templates on the lyso-
zyme nucleation were observed. Graphite templates induced an
average 60% reduction of the induction time in the droplets than
those without templates. Lysozyme tended to nucleate on the edge
of graphite flakes than on the top and bottom flat surfaces, due to
the roughness of the edge and the different CeC bonds on the edge
compared to the flat surface. With the graphene oxide templates,
the induction time reduced in the solution with high lysozyme
concentrations, but increased with low lysozyme concentrations.
With equal concentration of the salt and different concentrations of
lysozyme in the solution, the graphene oxide templates hindered
the nucleation, except with 1.1 M salts in the solution. The crystals
obtained with graphite and graphene oxide templates had similar
tetragonal shape as those without templates. However, the gra-
phene oxide templates were enclosed inside the crystal or lead to
rough and potholed crystal surface during the slow growth process.
In most of the equal conditions, crystal numbers in the droplets
followed the order: with graphite template > with graphene oxide
templates > without templates and the size of the crystals had the
opposite order. The various behaviours of crystallization process
could be due to the ionic strength, pH and electrostatic interactions
between templates and lysozyme molecules, which could be
further investigated by molecular simulations for better under-
standing the mechanism and potential applications in design the
selective crystallization templates, functionalize the graphite or
control the crystallization process.
33
Particuology 79 (2023) 27e34
Declaration of competing interest
There are no conflicts of interest.
Acknowledgements
Z. Lin and W. Tian would like to acknowledge school of AACME,
Loughborough University, and V. Yadav and H. Yang are grateful to
the UK EPSRC (Engineering and Physical Sciences Research Council)
for support (EP/T005378/1). The authors acknowledge use of the
facilities and the assistance of Sam Davis in the Loughborough
Materials Characterisation Centre.
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